Biotin is an essential protein cofactor used in carboxylation reactions central to human metabolism including enzymes involved in fatty acid biosynthesis, gluconeogenesis, and branched-chain amino acid catabolism. The terminal step in biotin biosynthesis involves the insertion of a sulfur atom between C6 and C9 of the precursor dethiobiotin, forming the biotin thioether ring. This insertion reaction is deceptively simple yet represents an impressive feat of enzymatic catalysis, requiring the enzyme break two saturated, inactivated CH bonds in dethiobiotin prior to sulfur insertion. This reaction is catalyzed by the E. Coli BioB protein, a dimeric iron- sulfur protein, and requires the participation of adenosylmethionine (AdoMet) and reduced flavodoxin. The requirement for AdoMet and flavodoxin suggests that biotin synthase is a member of a family of enzymes that reductively cleave AdoMet to generate a 5' - deoxyadenosyl radical, which is then used to generate a protein radical or to directly abstract a hydrogen atom from the substrate. More generally, biotin synthase belongs to a class of enzymes that are able to generate and control carbon and/or sulfur radicals in order to carry out difficult biosynthetic transformations; these enzymes include the human enzymes ribonucleotide reductase and lipoic acid synthase. Several obstacles have hindered in depth studies of the mechanism of biotin thioether ring formation: (I) the sulfur- containing substrate for BioB is not known, (II) the redox requirements for thioether ring formation are not known, and (III) the role of AdoMet and flavodoxin in enzyme activation is not understood. Studies described in this proposal address these substrate, protein, and activation requirements for biotin thioether ring formation, and will lead to more detailed studies of the chemical mechanism of radical-mediated sulfur insertion. By integrating information from these studies our knowledge of other enzymes in the biotin biosynthetic pathway, reconstitution of essential components of this pathway may in the future provide a convenient and inexpensive source of biotin for nutritional, therapeutic, and industrial applications.